Abstract
As determined by a guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)- 1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT2C (h5-HT2C) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1–2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI),d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT2C receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [35S]GTPγS binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi3, DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT2C receptors inasmuch as the selective 5-HT2C antagonist SB242,084 blocked [35S]GTPγS binding at both Gq/11 and Gi3. Agonist potency for stimulation of Gi3 was ∼6- to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT2C receptors. Inactivation of h5-HT2C receptors with the alkylating agentN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi3, indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi3 by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT2C receptors couple to both Gq/11 and Gi3in CHO cells, and efficacy for G protein subtype activation is both ligand- and receptor reserve-dependent.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
Log in using your username and password
Purchase access
In this issue
Differential Activation of Gq/11 and Gi3 Proteins at 5-Hydroxytryptamine2C Receptors Revealed by Antibody Capture Assays: Influence of Receptor Reserve and Relationship to Agonist-Directed Trafficking
